In the last decade, molecular biology has made a major impact in the understanding of normal cell function and alterations that take place in disease. The current technologies require the extraction of DNA or RNA from cells prior to analysis. In heterogeneous populations of cells, the actual cells that express a particular gene or have a particular gene amplified or mutated or translocated cannot be identified. In situ hybridization of genetic probes to intact cells or chromosomes fixed to microscope slides has been successfully performed to measure genetic parameters on an individual cell or chromosome basis. This is disclosed by Singer et al. in Biotechniques 4:230, 1986; Singer et al. in Proc. National Acad. Sci. 79:7331-7335, (1982); and Bakkus et al. in Oncogene 4:1255-1262, (1989).
The polymerase chain reaction (PCR) is extensively used to amplify genomic DNA extracted from cells to provide genetic material for further study. This has been disclosed by Saiki et al. in Science 239:487 (1988); by Frye in Oncogene 4: 1153-1157 (1989); by Saiki et al. in Nature, 324, 163-165 (1986); by Almoguera et al. in Cell 549-554 (1988); and Mullis et al. in Methods in Enzymology 155: 335-351 (1987). The PCR is also disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures of which are incorporated herein by reference. More recently RNA has been studied by first using reverse transcriptase to convert RNA into DNA and then to amplify the DNA using the PCR as disclosed by Schriever et al. in J. Exp. Med. 169:2043-2058 (1989) and Kawasaki et al. in Proc. Nat'l. Acad. Sci USA 85:5698-5702 (1988). These procedures utilize RNA or DNA that is first extracted from intact cells. Further manipulation takes place in a cell free environment wherein all reaction substances are in solution. This technology has also been applied to the amplification of viral RNA using a reverse transcriptase to make a cDNA as disclosed by Feorino et al. in Science 225:69-72 (1984) and Coombs et al. at NEJM 321:1626-1631 (1989). Tecott, et al. disclose in Science 240:1661-1664 (1988) that they have carried out the reverse transcriptase step in paraformaldehyde fixed intact cells using a radioactive nucleotide for detection. Frye, et al. in Oncogene 4:1153-1157 (1989) insinuated that they performed PCR on intact cells but they provided no methodology or data for performing this task. Since their evidence for a reaction product was standard gel electrophoresis, it can be concluded they did not actually perform the PCR in the cells themselves. Bauman, et al. in Acta Histochemica Suppl. 37:65-69 (1989) suggested that Flow Cytometry would provide several advantages for measuring specific RNA and DNA sequences, and provide examples using in situ hybridization but they did not use the polymerase chain reaction. Finally, Haase et al. in Proc. Nat'l. Acad. Sci. USA, Vol. 87, pp 4971-4975, in Jul. 1990, disclosed conducting the PCR inside certain cells, but did not suggest measuring the cells with flow cytometry. The forgoing references are incorporated herein by reference as background information.